Methods for improved diagnosis of dysplasias

ABSTRACT

The present invention relates to a method for improved diagnosis of dysplasias based on simultaneous detection of INK4a gene products and at least one marker for cell proliferation. Particularly the present invention provides a method for discriminating dysplastic cells over-expressing INK4a gene products from cells over-expressing INK4a gene products without being dysplastic by detection of a marker suitable for characterising the proliferation properties of the respective cell. The characterisation of the proliferation properties may comprise the detection of a marker or a set of markers characteristic for active cell proliferation and/or a marker or a set of markers characteristic for retarded or ceased cell proliferation. The method presented herein thus enables for a specific diagnosis of dysplasias in histological and cytological specimens.

This application is a continuation application of U.S. patentapplication Ser. No. 10/533,384, which is a National Stage ofInternational Application PCT/EP03/050738, filed Oct. 21, 2003,published May 6, 2004, under PCT Article 21(2) in English; which claimsthe priority of EP 02024030.5, filed Oct. 28, 2002; and EP 03100584.6,filed Mar. 7, 2003.

FIELD OF THE INVENTION

The present invention relates to a method for improved diagnosis ofdysplasias based on simultaneous detection of INK4a gene products and atleast one marker for cell proliferation. In particular, the presentinvention provides a method for discriminating dysplastic cellsover-expressing INK4a gene products from cells over-expressing INK4agene products without being dysplastic by detection of a marker suitablefor characterising the proliferation properties of the respective cell.The characterisation of the proliferation properties may comprise thedetection of a marker or a set of markers characteristic for active cellproliferation and/or a marker or a set of markers characteristic forretarded or ceased cell proliferation. The method presented herein thusenables a specific diagnosis of dysplasias in histological andcytological specimens. The detection of the over-expression ofp16^(INK4a) in biological samples has proven as a useful marker in thedetection of anogenital lesions, such as carcinoma of the uterine cervix(see WO00/01845; Klaes et al., Int. J. Cancer: 92, 276-284 (2001)). Themethod based on p16^(INK4a)-specific immuno-chemical staining allows fora sensitive and specific identification of dysplastic cells in tissuesections and in cytological samples.

In immuno-histochemical examinations of tissues, dysplastic andneoplastic cells can be stained using a p16^(INK4a) specific antibodymediated staining procedure. The histological diagnosis of neoplasticlesions can thus be supported by a staining procedure based on amolecular marker characteristic for transformation of cells inanogenital lesions. The diagnosis, whether or not cells are neoplastic,in these procedures is not solely based on the p16^(INK4a) specificstaining, but does also rely on the histological information.

This is due to the fact that, in about 20-30% of samples, metaplasticcells show some immunoreactivity with p16^(INK4a) specific antibodies,and thus are stained in the course of the predominantly less than thatof neoplastic cells.

The common methods used in screening tests for the early detection ofdysplasias and/or neoplasias do not employ histology based tests, butrather rely on cytological testing procedures. Yet especially in caseswhen there is no histological information available concerning thearchitecture of tissues, such as, for example, in cytologicalexaminations, testing for p16^(INK4a) over-expression alone may lead tofalse positive results. This is due to the fact that those fractions ofmetaplastic cells expressing p16^(INK4a) at detectably elevated levelsmay not be differentiated by means of a histologic criteria.

The percentage of cells showing over-expression of p16^(INK4a) increasesin the course of emergence of dysplasias. So, in neoplastic orpre-neoplastic stages, when only a restricted population of neoplasticor pre-neoplastic cells is present in samples, the immunoreactivity ofp16^(INK4a) may be weak. This weak immunoreactivity may be of about thelevel as the level caused by metaplastic cells. In later stages ofdysplasias, the overall immunoreactivity of p16^(INK4a) is stronger, soneoplastic lesions are easily discernible from metaplasias even in acytological testing format. This might lead to cases where the presenceof metaplastic cells expressing p16^(INK4a) might be confused with thepresence of neoplastic cells, and thus produce a false positive result.Especially in screening tests, where the detection of early stages ofneoplasias is desirable, this condition is quite unpleasant. This isespecially true, as the p16^(INK4a) based diagnosis has proven to be avaluable tool in histological examinations and the application incytological based screening procedures would be able to enhance theseestablished procedures. To reduce false positive results in cytologicaltesting formats and thereby further enhance the fidelity of thep16^(INK4a) mediated diagnosis of anogenital lesions, a method fordiscriminating the metaplasias from neoplastic and dysplastic lesionswould be desirable. A method for the discrimination of metaplasias fromneoplastic and pre-neoplastic lesions is provided within the embodimentsclaimed according to the present invention.

For supporting the discrimination of metaplasias from neoplastic lesionsin testing procedures based on the over-expression of p16^(INK4a), amarker molecule would be desirable that is expressed in neoplasticand/or pre-neoplastic cells and tissues and which is not expressedsimultaneously with INK4a gene products in one single metaplastic cell.

A solution to the problem present in the art is provided by the methodsclaimed according to this invention. In the course of the experimentsleading to the present invention, the inventors have found that acombination of detection of the presence or absence and/or the level ofp16^(INK4a), in combination with at least one marker for cellproliferation, such as e.g. Ki67, Ki-S2, mcm5 or mcm2, may solve theproblem present in the art.

In the art, a couple of documents pertaining to the use of a combinationof molecular markers for improved diagnosis of dysplasias is presented.In WO0208764, a method for improved diagnosis of cervical malignanciesis disclosed using a combination of an HPV marker and a marker for cellproliferation or viral activity. p16^(INK4a) is mentioned in the contextof this invention as a viral activity marker to be combined with HPVmarkers.

In EP1217377, a method for automated detection of cervical malignanciesis disclosed that is mediated by detection of more than one markermolecule. Some defined marker combinations are named within thedocument. There is no disclosure in this document relating to the choiceof suitable markers for a combination. The purpose of the combination inthis application is improved fidelity of the automated analysis ofstaining patterns in biological cytological specimens. This documentmentions combination of p16^(INK4a) with other tumor markers. WO02059616discloses a method for detection of cell-cycle disturbances for improveddiagnosis of cervical malignancies. The document discloses thatdysplastic cells exhibit disturbances in cell cycle control, and maythus be identified by means of detection of cyclin E type proteinstogether with post G1 substances in cells.

Jeffrey Keating in “Ki67, Cyclin E, and p16^(INK4a) Are ComplimentarySurrogate Biomarkers for human papilloma Virus-Related CervicalNeoplasia” (American Journal of Surgical Pathology 25(7): 884-891,(2001)) discloses the complementarity of the use of p16^(INK4a), Ki67,and Cyclin E in the course of diagnosis of cervical dysplasias. Thedocument refers to the problems of each single marker in the detectionof dysplasias, and states that p16^(INK4a) in combination with Cyclin Emay be suitable to overcome the drawbacks of the single marker molecule,especially when cytological specimens are under examination. Nodisclosure teaching use of p16^(INK4a) in concert with a markercharacteristic of proliferating cells such as Ki67 is given. Thedocument does not teach the combination of p16^(INK4a) for use indiagnostic methods; moreover, the disclosure pertains to a restricteduse of Ki67 in cervical differential diagnosis, and thus, teaches awayfrom the use of this marker in diagnosis of cervical malignancies.

In the art, there is no disclosure teaching the use of a combination ofp16^(INK4a) with a marker characteristic of cell proliferation for usein a diagnostic method for improved discrimination of p16^(INK4a)positive non-dysplastic cells from p16^(INK4a) positive dysplasias.WO02059616 does not give a hint as to the use of p16^(INK4a) in thedetection of dysplastic cell proliferation. EP1217377 does not teach apurpose for the combination of tumor markers in the course of thedetection other than the automation of the analysis process. There is nodisclosure pertaining to the advantage of combinations for specificdiscrimination purposes such as discrimination of dysplastic cells frome.g. metaplastic cells in cervical specimens.

SUMMARY OF THE INVENTION

The inventors of the present invention sought to overcome the drawbackpresent in the art that p16^(INK4a), over-expressed in variousdysplasias, may also be detected in some other non-dysplastic cells. Thediscrimination between non-dysplastic p16^(INK4a) positive cells anddysplastic cells over-expressing p16^(INK4a) may be based on theproliferation characteristics of the respective cells. In normal cells,p16^(INK4a) inhibits cdk4, and thus inhibits proliferation. In contrast,in dysplastic cells, this regulation is impaired. Thus, p16^(INK4a) doesnot lead—despite its uncommonly high expression level—to an inhibitionof the cell proliferation.

The inventors of the present invention found that dysplastic cells maybe discriminated from cells exhibiting controlled cell proliferation bythe simultaneous detection of p16^(INK4a) with a marker characteristicfor cell proliferation. Due to the fact that, in normal cells, elevatedlevels of p16^(INK4a) inhibit cell proliferation, cells over-expressingp16^(INK4a) may be classified as being dysplastic provided they areexhibiting the characteristics of active cell proliferation.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withdrawing(s) will be provided by the Office upon request and payment ofthe necessary fee.

FIG. 1 is an example of fluorescent staining of a histological specimenof the cervix uteri. FIG. 1 shows staining of a severe dysplasia usingantibodies directed against p16^(INK4a). For experimental details, seeExample 1. Immunoreactivity for p16^(INK4a) renders green fluorescence.Almost all cells of the lesion are diffusely positively stained in thecytoplasm and in the nuclei.

FIG. 2 is an example of fluorescent staining of a histological specimenof the cervix uteri. FIG. 2 shows staining of a severe dysplasia usingantibodies directed against Ki67. For experimental details, seeExample 1. Immunoreactivity for Ki67 renders red fluorescence. Manycells of the dysplasia show nuclear positivity for Ki67.

FIG. 3 is an example of fluorescent double staining of a histologicalspecimen of the cervix uteri. FIG. 3 shows staining of a severedysplasia using antibodies directed against Ki67 and those directedagainst p16^(INK4a). For experimental details, see Example 1.Immunoreactivity for Ki67 renders red fluorescence, immunoreactivity forp16^(INK4a) renders green fluorescence and the overlay of red and greenfluorescence renders yellow fluorescence. Many cells of the dysplasiashow nuclear positivity for Ki67, as well as positivity for p16^(INK4a),and thus give rise to yellow fluorescence.

FIG. 4 is an example of fluorescent staining of a histological specimenof the cervix uteri. FIG. 4 shows staining of a squamous metaplasiausing antibodies directed against p16^(INK4a). For experimental details,see Example 1. Immunoreactivity for p16^(INK4a) renders greenfluorescence. Some cells of the metaplasia stain diffusely positive inthe cytoplasm and in the nuclei.

FIG. 5 is an example of fluorescent staining of a histological specimenof the cervix uteri. FIG. 5 shows staining of a squamous metaplasiausing antibodies directed against Ki67. For experimental details, seeExample 1. Immunoreactivity for Ki67 renders red fluorescence. Manycells of the squamous metaplasia show nuclear positivity for Ki67. Areasthat have been identified as positively expressing p16^(INK4a) do notshow any positive staining for Ki67, indicating lack of expression ofthe antigen in these areas.

FIG. 6 is an example of fluorescent double staining of a histologicalspecimen of the cervix uteri. FIG. 6 shows staining of a squamousmetaplasia using antibodies directed against Ki67 and againstp16^(INK4a). For experimental details, see Example 1. Immunoreactivityfor Ki67 renders red fluorescence, immunoreactivity for p16^(INK4a)renders green fluorescence and the overlay of red and green fluorescencerenders yellow fluorescence. Areas that are positively expressingp16^(INK4a) do not show any positive staining for Ki67, indicating lackof expression of the antigen in these areas. No cells in the specimengive rise to yellow fluorescence.

FIG. 7 is an example of chromogenic double staining of a histologicalspecimen of the cervix uteri. FIG. 7 shows staining of a severedysplasia using antibodies directed against Ki67 and againstp16^(INK4a). For experimental details, see Example 6. Immunoreactivityfor Ki67 renders red nuclear staining, immunoreactivity for p16^(INK4a)renders brownish staining over the whole cell, and double stainingrenders brown cells with red nuclei. Many cells of the dysplasia shownuclear positivity for Ki67, as well as positivity for p16^(INK4a), andthus, give rise to a pattern of brown cells with red nuclei.

FIG. 8 is an example of chromogenic double staining of a cytologicalspecimen of the cervix uteri. FIG. 8 shows staining of a severedysplasia using antibodies directed against Ki67 and againstp16^(INK4a). For experimental details, see Example 7. Immunoreactivityfor Ki67 renders red nuclear staining, immunoreactivity for p16^(INK4a)renders brownish staining over the whole cell, and double stainingrenders brown cells with red nuclei. Many cells of the dysplasia shownuclear positivity for Ki67 as well as positivity for p16^(INK4a), andthus, give rise to a pattern of brown cells with red nuclei.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for discrimination ofneoplastic, pre-neoplastic and/or dysplastic lesions from non-dysplasticcells showing elevated levels of p16^(INK4a), in biological samples in ahistological or cytological testing procedure based on the detection ofthe presence or absence of cells expressing p16^(INK4a) genesimultaneously with cell proliferation markers in said biologicalsamples. Suitable markers for cell proliferation may be, e.g., Ki67,Ki-S2, Ki-S5, mcm5, or mcm2. In one embodiment of the invention, proteinor mRNA of the markers for cell proliferation may serve as a marker fordiscrimination of metaplasias from early dysplastic or pre-neoplasticlesions in samples. One aspect of the present invention is to provide amethod for discriminating dysplastic cells over-expressing INK4a geneproducts from other cells expressing INK4a gene products at a detectablelevel in biological samples comprising determining in a cytological orhistological testing procedure the co-expression of at least two markermolecules in at least one single cell, wherein at least one markermolecule is an expression product encoded by the INK4a gene and at leastone further marker molecule is a cell proliferation marker, wherein theover-expression of at least one INK4a gene product and expression of atleast one marker for active cell proliferation at an immuno-chemicallydetectable level within said single cell is indicative of the dysplasticstate of the cell, and wherein the over-expression of at least one INK4agene product and expression of at least one marker for senescence,terminal differentiation of cells, apoptosis, or cell cycle arrest at adetectable level within said single cell is indicative of thenon-dysplastic state of the cell. A second aspect of the presentinvention relates to a test kit for determination of dysplasias insamples according to the method disclosed herein.

During the experiments leading to the present invention, it was foundthat, under certain circumstances, non-dysplastic cells may showimmunoreactivity for p16^(INK4a). The inventors found that these cellsexhibiting ordered control of cell proliferation in response to theelevated levels of the INK4a gene products are subject to growth arrest.Thus, these individual cells do not show immunoreactivity for markers ofcell proliferation. In contrast, transformed cells over-expressingp16^(INK4a) exhibit dysregulated control of cell proliferation, and donot respond to the elevated level of p16^(INK4a) by cessation ofproliferation. Thus, these dysplastic cell show simultaneous expressionof p16^(INK4a) with markers for cell proliferation. The inventors thusfound that a simultaneous detection of p16^(INK4a) with markers for cellproliferation may serve to discriminate dysplastic cells from arrestedcells over-expressing p16^(INK4a) such as, e.g., metaplastic cells.

The discovery that p16^(INK4a) over-expressed in various dysplasias mayalso be detected in some other non-dysplastic cells led the inventors ofthe presented method to establish a technique for discrimination betweennon-dysplastic p16^(INK4a) positive cells and dysplastic cellsover-expressing p16^(INK4a) based on the proliferation characteristicsof the respective cells. Whereas in normal cells p16^(INK4a) inhibitscdk4, and thus inhibits cell proliferation, this is not true fordysplastic cells. Thus, in dysplastic cells p16^(INK4a) does notlead—despite it's uncommonly high expression level—to an inhibition ofthe cell proliferation.

The method disclosed herein is based on the fact that dysplastic cellsmay be discriminated from cells exhibiting normal cell proliferation bythe simultaneous detection of an INK4a gene product such as, e.g.,p16^(INK4a), with a marker characteristic for cell proliferation. Theterm marker, as well as marker molecules in general, shall be usedherein to pertain to proliferation marker gene expression products aswell as to INK4a gene expression products. The denominations giventhroughout this text for genes may, in part, relate to the genes orproteins as they have been discovered from any organism. In the contextof the present invention, this denomination shall confer to therespective homologue of the named markers in the organism which isparticularly in question for a method as disclosed herein. In certainembodiments of the present invention, this organism is a mammal and, inone embodiment, may be a human being. Thus, in one embodiment of thepresent invention, the named markers shall be the human homologues ofthe respective denominated ones.

Generally throughout the text, the term “(cell) proliferation marker” or“marker for cell proliferation” in the various grammatical forms is usedto denominate proteins as well as nucleic acid markers. In case theprotein name of a marker such as e.g. “replication protein” is usedherein, this use shall be understood metonymically and pertain as wellto the protein as to the nucleic acid marker molecules encoding theparticular protein

A marker useful according to the present invention may be any moleculetranscribed from a gene or any molecule translated from such atranscript. Thus, “gene product”, as used in the context of the presentinvention, may comprise polynucleotides such as, e.g., DNA or RNA andpolypeptides such as proteins, proteoglycans, peptides, etc. “Expressionproduct(s)” as used in the context of the present invention shallcomprise any transcript of a gene locus in forward or reverse directionincluding any reading frames and splicing variants. “Expressionproducts” as used herein shall thus comprise any alternative productsencoded by the nucleic acids of a particular gene locus. INK4a encodedgene-products, as used in the context of the present invention, shall beany mRNA transcribed from the INK4a gene locus or any polypeptidetranslated from such an mRNA. In one embodiment of the invention, theexpression products encoded by the INK4a gene may exhibit molecularweights of about 5 to 40 kDa or any value in between, and preferably ofabout 10 to 20 kDa or any value in between, and most preferably of about14 to about 19 kDa or any value in between, respectively.

INK4a gene-products suitable for the method according to the presentinvention may comprise, e.g., gene-products such as, e.g., p16^(INK4a)and p14ARF. The term “(cell) proliferation marker” or “marker for cellproliferation”, as used in the context of the present invention, shallcomprise any marker molecule known in the art to be characteristic forthe proliferation status of cells. The proliferation status may be,e.g., a status of actively proliferating cells, of retarded cellproliferation, of arrested cell proliferation, of senescent cells, ofterminally differentiated cells, of apoptosis, etc. In one embodiment ofthe invention, the cell proliferation marker is a marker moleculecharacteristic for active cell proliferation. In another embodiment ofthe invention, the proliferation marker molecule may be a moleculecharacteristic for arrested, terminally differentiated, senescent, orapoptotic cells.

In certain embodiments, proliferation markers for use in the context ofthe present invention may comprise genes engaged in the DNA replication,such as e.g., proteins of the pre-initiation complex or of thereplication fork. Such molecules may comprise, e.g., helicases, such aseucaryotic helicase or MCM proteins (MCM2, MCM3, MCM4, MCM5, MCM6,MCM7), protein TP as disclosed in WO0050451 and WO0217947 (alsodenominated HELAD1, Pomfil2, Unc-53), kinases or phosphatases engaged inthe replication process such as, e.g., CDC6, CDC7 protein kinase, Dbf4,CDC14 protein phosphatase, CDC45 and MCM10. Furthermore, proliferationmarkers may comprise proteins engaged in the processive replication forksuch as, e.g., PCNA or DNA polymerase delta, replication protein A(RPA), replication factor C (RFC), and FEN1.

In other embodiments, the proliferation markers may comprise moleculesnecessary for the maintenance of cell proliferation such as Ki67, Ki-S5,or Ki-S2. In this embodiment, proteins may be present, e.g., throughoutthe whole cell cycle. They are useful for performing a method accordingto the present invention provided they are characteristic of active cellproliferation and are not significantly expressed in arrested,terminally differentiated, apoptotic, or senescent states of cells.Ki67, Ki-S2, and Ki-S5 as used herein shall denominate the proteinmarker molecules detected by the respective antibodies as well as thenucleic acids encoding these antigens.

In another embodiment, the cell proliferation markers for use in amethod according to the present invention may be a marker moleculecharacteristic of retarded or ceased cell proliferation such as, e.g., asenescence marker, a cell cycle arrest marker, a marker characteristicfor terminally differentiated cells, or an apoptosis marker. Suchmolecules comprise, e.g., p21, p27, Caspases, BAD, CD95, fas-ligand,parp-proteins, etc.

Discrimination as used in the context of the present invention shallcomprise an assessment whether a sample is to be classified in one oranother way. In one embodiment of the invention, the discriminationpertains to the assessment of a tissue, or components thereof, beingdysplastic or being not dysplastic. Thus, the discrimination as usedherein is a judgement about the growth properties of cells in abiological sample.

In one embodiment of the present invention, the discrimination comprisesthe detection of expression of an INK4a gene product simultaneously withthe detection of expression of a marker characteristic for active cellproliferation. In this case, cells co-expressing both marker moleculesare to be classified as being dysplastic.

In another embodiment of the present invention, the discriminationcomprises the detection of an INK4a gene product simultaneously with thedetection of expression of a marker characteristic for arrested, ceased,or retarded cell proliferation. In this case, cells co-expressing bothmarker molecules are to be classified as being non-dysplastic.

In certain embodiments of the present invention, it will be useful todetect the presence or absence and/or the level of more than two markermolecules. In one embodiment, one INK4a gene expression product will bedetected in concert with two or more markers for cell proliferation.This may be useful to enhance the identification of proliferationproperties of the INK4a gene product-expressing cells in samples. Someproliferation markers are restricted to specific phases of the cellcycle or are present in low abundance in cells. Due to this fact, insome cases, the detection of proliferating cells expressing INK4a geneproducts may be improved by detection of two or more proliferationmarkers. In such cases, e.g., one proliferation being expressed duringthe whole proliferative cell cycle may be detected simultaneously bymarkers characteristic for specific cells cycle phases. For example,Ki67, Ki-S5, or Ki-S2 may be detected together with mcm5, mcm2, PCNA,rpA, rfC, etc. In other cases, e.g., proteins engaged in the DNAreplication may be detected together with Ki67, Ki-S5, or Ki-S2. In yetanother case, Ki67 may be detected together with Ki-S2. It must beunderstood that these examples are intended to exemplify combinatorialpossibilities, and shall not be comprehensive, so that various othercombinations of proliferation markers are likewise useful and suitablein the procedure of the method according to the present invention. Asthe case may be, combination of two or more cell proliferation markermolecules may be applied in a method as disclosed herein. In anotherembodiment, two or more marker molecules detectable over largerstretches of the cell cycle or even over the whole cell cycle, or in anactively proliferating cell, may be detected in concert in a method asdisclosed herein. A combination of more than one cell proliferationmarker molecules may be useful generally for improving the sensitivityof the detection of the proliferation characteristics of cells.

In certain embodiments of the present invention, a combination mayfurthermore comprise other marker molecules such as senescence markermolecules, markers for arrested cells, markers for terminallydifferentiated cells, markers for apoptotic cells, markers for viralinfection or for viral activity in cells, or cell cycle regulatoryprotein markers. In certain embodiments, in connection with dysplasiasbeing associated with HPV infection, a detection of HPV associatedmarker molecules or markers for viral activity may be of use for adetection of a dysplasia. The methods useful for detection of HPVinfection in samples are known to those of skill in the art. Thesemethods may comprise methods employing probes specific for HPV agents ormay employ nucleic acid amplification reactions. The detection of aviral infection may be carried out simultaneously or subsequently to thedetection of the INK4a and proliferation marker molecules.

Dysplastic, as used in the context of the present invention, shall referto dysplasias from mild to severe dysplasias and their precursorystages, as well as carcinoma such as carcinomas in situ or invasivecarcinomas and disseminated tumor cells. Thus, dysplastic as used hereinshall also comprise early and precursory stages of dysplasias andcarcinomas. Cells over-expressing INK4a gene products without beingdysplastic (non-dysplastic as used herein) as mentioned herein maycomprise, e.g., metaplastic cells, senescent cells, terminallydifferentiated cells, or cells that in certain stages of the cell cycleexhibit elevated levels of the INK4a gene products. In certain cells,elevated levels of INK4a gene products may even be effected as aresponse to external signals such as hormones, transmitters, etc. In oneembodiment of the present invention, the non-dysplastic cellsover-expressing INK4a gene products comprise, e.g., metaplastic cells,endometrial cells, etc.

The method for detection of the expression level of the INK4a encodedgene-products and/or the proliferation marker gene products according tothe present invention is any method, which may (but need not) be, e.g.,suited to detect even very small amounts of specific biologicalmolecules in biological samples. The detection reaction according to thepresent invention is a detection either on the level of nucleic acids oron the level of polypeptides.

A marker molecule is said to be detectable as used in the context of thepresent invention, provided the marker may be detected in the course ofsuitable detection procedure such as, e.g., in situ hybridization,immuno-chemical staining, hybrid capture assay, etc. The level ofexpression of a marker molecule may be made detectable using suitablereporter reactions such as, e.g., a chromogenic or fluorescence basedimmuno-chemical staining or in-situ-hybridization procedure formicroscopic or automated analysis. Suitable methods for enhancing thereporter signal known to those of skill in the art may be applied in thecourse of a method according to the present invention. Thus, the markeris said to be detectable in a case where the staining supersedes therespective background staining inherently obtained in theimmuno-chemical staining procedure so as to produce significant stainingresults.

The marker molecules may be detected using reagents that specificallyrecognize these molecules. The detection reaction for the INK4agene-products and/or the proliferation marker gene products may compriseone or more reactions with detecting agents either recognizing theinitial marker molecules or recognizing the prior molecules used torecognize other molecules. In certain embodiments of the presentinvention, two or more probes may be used for the detection of onesingle marker molecule. For example, two or more different bindingagents (e.g., antibodies) or oligonucleotide probes directed against onesingle marker molecule (or, as the case may be, against differentepitopes or different sequences) may be used in the course of the methodas disclosed herein.

The detection of the different gene products may be performed in onereaction vessel or container or in different containers simultaneouslyor subsequently in time. Thus, the different gene products may bedetected simultaneously in one cell co-expressing both products.Otherwise, cells co-expressing the gene products may be used forseparated detection reaction (separated in space or in time) to detecteach a single marker in the cells. In another embodiment, there might becells expressing one or the other marker. The detection of the markermolecules in the different cells may also be performed simultaneously orseparately in time and/or space.

The detection reaction further may comprise a reporter reactionindicating the presence or absence and/or the level of the markermolecule gene-products. The reporter reaction may be, for example, areaction producing a coloured compound, a bioluminescence reaction, afluorescence reaction, generally a radiation emitting reaction, etc.

In certain embodiments, different marker molecules may be recognized byagents that produce different reporter signals, so that the signalsreferring to marker molecules can be distinguished. In one preferredembodiment of the invention, the detection of the expression of the twoor more INK4a gene-products and/or proliferation marker gene products iscarried out simultaneously. In this case, the reporter reaction mayemploy, for example, different fluorescent labels for the differentmolecules detected.

However, within the context of the present invention, it must notnecessarily be answered whether the one or the other proliferationmarker or INK4a marker gene product is expressed in the cells. Incertain embodiments, the question is whether any proliferation markerand/or INK4a gene product is expressed. In the course of theexperiments, a procedure may be chosen that gives the same fluorescencesignal as an indication of the presence of a proliferation marker. Thisprocedure is suitable to improve sensitivity of the detection of thecell proliferation characteristics (different markers characteristic foractive cell proliferation). As the case may be, the procedure may beapplied so as to render one detectable signal for three, four, or evenmore marker molecules characteristic of cell proliferation. Analogously,the same may under certain circumstances be true for the INK4a geneexpression products. It must be understood that different stainingsignals for different proliferation marker molecules may be desirable.The procedures may be applied to the necessities of the respectiveexperiment.

In certain embodiments of the present invention, a combination of one ormore (e.g., two different) INK4a gene products may be detected with acombination of one or more (e.g., a set of two, a set of three, a set offour, a set of five or a set of even more) markers for cellsproliferation. In some cases, the detection of the marker molecules forcell proliferation may render only one reporter signal. In other cases,each single marker for cell proliferation may render a specific reportersignal or groups of marker molecules may render specific reportersignals.

Signals for the indication of the presence of immunoreactivity may bechromogenic signals produced by various methods known in the art.Alternatively, or even in combination, fluorescent signals may be used.Suitable reporter signals comprise fluorescent labels such asfluorescein, rhodamine, etc.

Applicable formats for the detection reaction according to the presentinvention may be blotting techniques, such as Western-Blot,Southern-blot, Northern-blot, and immunocytochemical, orimmunohistochemical procedures. The blotting techniques are known tothose of ordinary skill in the art, and may be performed as, for exampleelectro-blots, semidry-blots, vacuum-blots or dot-blots.Immunocyto/histochemical staining procedures are known to those of skillin the art, and may comprise binding agent-mediated detection ofpolypeptides as well as in situ hybridisation techniques. Both differenttechniques may even be applied simultaneously. In certain embodiments,hybrid capture of nucleic acids may be used for the detection.Amplification reactions may also be applicable for the detection of e.g.nucleic acid molecules.

In one embodiment of the invention, the detection of the level of INK4aand/or proliferation marker gene-products is carried out by detection ofthe respective nucleic acids (e.g., mRNA) or fragments thereof presentin the sample. The means for detection of nucleic acid molecules areknown to those skilled in the art. The procedure for the detection ofnucleic acids can be carried out by, for example, a binding reaction ofthe molecule to be detected to complementary nucleic acid probes,proteins with binding specificity for the nucleic acids, or any otherentities specifically recognizing and binding to said nucleic acids. Inone embodiment, in situ hybridisation of oligonucleotide probes tonucleic acids in a sample may be used for the detection of expressionproducts or markers. A probe, as used in the context of the presentinvention, may be any agent binding specifically to a molecule. In thecase of nucleic acids, a probe may be an oligonucleotide hybridising toa particular sequence. In one embodiment, the probe may be, e.g., aprimer. In the case of the detection of polypeptides or proteins, theprobe as used herein may be, e.g., a binding agent such as an antibody.In certain embodiments of the present invention, the probes may bedetectably labelled. The label may be selected from the group comprisinga radioisotope, a bioluminescent compound, a chemiluminescent compound,a fluorescent compound, a metal chelate, or an enzyme. Probes may beapplied in any detection procedure known in the art as, for example, inthe course of an in situ hybridisation procedure, in the course ofhybrid capture assays, in the course of immuno-chemical stainingreaction, in the course of blotting techniques, etc.

This method may be performed as well in vitro as directly in situ forexample, in the course of a detecting staining reaction. Another way ofdetecting the marker mRNAs in a sample performed in the method accordingto the present invention is an amplification reaction of nucleic acids,which can be carried out in a quantitative manner such as, for example,the polymerase chain reaction. In a preferred embodiment of the presentinvention, real time (RT) PCR may be used to quantify the level ofmarker mRNAs in samples of dysplasias or tumors (cells or tissuesamples).

In another preferred embodiment of the invention, the detection of thelevel of INK4a and/or proliferation marker gene-products is carried outby determining the level of expression of a protein or fragmentsthereof. The determination of the marker gene-product on the proteinlevel can, for example, be carried out in a reaction comprising abinding agent specific for the detection of the particular markerpolypeptide.

The binding agents can be used in many different detection techniquesincluding, for example, in western-blot, ELISA or immuno-precipitation.Generally, polypeptide binding agent based detection can be carried outas well in vitro as directly in situ as, for example, in the course ofan immuno-chemical staining reaction. Any other method for determiningthe amount of particular polypeptides in biological samples can be usedaccording to the present invention. The immuno-cytochemical imagingprocedures for use in the context of the present invention may comprise,e.g., the staining of cytological or histological preparations withchromogenic or fluorescent dyes. The staining may comprise, e.g.,binding of the molecules to be detected by a first binding agent, whichitself is detected by a secondary binding agent which may be labelled.The first binding agent may in certain embodiments be a nucleic acid ora protein binding agent (e.g. an antibody) and the secondary bindingagent may be, e.g., a secondary antibody recognizing the first bindingagent.

Any methods known in the art for performing staining of cytochemical orhistochemical staining may be applied in the course of a methodaccording to the present invention.

Binding agents as used in the context of the present invention for thedetection of the level of INK4a polypeptides such as p16^(INK4a) orp14ARF polypeptides and proliferation marker polypeptides such as, e.g.,mcm5, mcm2, Ki67, Ki-S5, PCNA, or Ki-S2 polypeptides may compriseantibodies and antigen-binding fragments, bi-functional hybridantibodies, peptidomimetics containing minimal antigen-binding epitopes,anti-cullines (Anti-Caline™), etc.

An antibody or antigen-binding agent is said to react specifically if itreacts at a detectable level with a protein disclosed herein, and doesnot significantly react with other proteins. The antibodies according tothe present invention may be monoclonal or polyclonal antibodies. Asused herein, the term antibody or monoclonal antibody is meant toinclude intact molecules as well as antibody fragments. Moreover,antibodies of the present invention include chimeric, single chain, andhumanized antibodies.

According to the present invention binding agents may be used isolatedor in combination. By means of combination, it is possible to achieve ahigher degree of sensitivity. The term antibody, preferably, relates toantibodies which consist essentially of pooled monoclonal antibodieswith different epitopic specificities, as well as distinct monoclonalantibody preparations. Monoclonal antibodies are made fromantigen-containing fragments of the polypeptide of the invention usingany of a variety of techniques known to those of ordinary skill in theart (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, 1988). In one such technique, an immunogencomprising the antigenic polypeptide, or a synthetic part thereof, isinitially injected into any of a wide variety of mammals (e.g., mice,rats, rabbits, sheep, and goats). In this step, the polypeptides of thisinvention may serve as the immunogen without modification.Alternatively, particularly for relatively short polypeptides, asuperior immune response may be elicited if the polypeptide is joined toa carrier protein, such as bovine serum albumin or keyhole limpethemocyanin. The immunogen is injected into the animal host, preferablyaccording to a predetermined schedule incorporating one or more boosterimmunizations, and the animals are bled periodically. Polyclonalantibodies specific for the polypeptide may then be purified from suchantisera by, for example, affinity chromatography using the polypeptidecoupled to a suitable solid support.

Within the context of the present invention, it must not necessarily beanswered whether one or the other proliferation marker is expressed inthe cells. In certain embodiments, the main question may be whether anyproliferation marker is expressed. In the course of the experiments, aprocedure was chosen that gives the same fluorescence signal as anindication of the presence of a proliferation marker. This procedure issuitable to improve sensitivity of the detection of the cellproliferation characteristics. As the case may be, the procedure may beapplied so as to render one detectable signal for three, four, or evenmore marker molecules characteristic for cell proliferation.Analogously, under certain circumstances, the same may be true for theINK4a gene expression products. it must be understood, that, as the casemay be, different staining signals for different proliferation markermolecules may be desirable. The procedures may be applied to thenecessities of the respective experiment.

The INK4a gene-products and/or proliferation marker gene products may,according to the present invention, be detected simultaneously. In thiscontext, simultaneously according to the present invention shall meaneither literally at the same instant or within the same testingprocedure, whereby the single detection steps are temporarilyconsecutive. The detection procedure according to the present inventionmay, furthermore, comprise a cytochemical staining procedure rendering achromogenic or fluorescent staining of cells or cell compartments. Suchstaining procedures are known to those of skill in the art and maycomprise, for example, staining for acidophilic or basophilicstructures, of subcellular regions (e.g. the nucleus, the mitochondria,the golgi, the cytoplasm, etc.), of specific molecules (the chormosomes,of lipids, of glycoproteins, of polysaccharids, etc.) in the cytologicalspecimens. Fluorescence dyes such as DAPI, Quinacrin, Chromomycin, etc.may be employed. Furthermore, chromogenic dyes such as Azan,Acridin-orange, Hematoxylin, Eosin, Sudan-red, and Thiazin—stains(Toluidin-blue, Thionin) may be applied. In other embodiments, stainingprocedures such as Pap-staining, Giemsa-staining, Hematoxylin-Eosinstaining, van-Gieson staining, Schiff-staining (using Schiff reagent),staining procedures employing precipitation of metals (such as, e.g., ofsilver in staining procedures employing Silver Nitrate), or insolublestains such as, e.g., of Turnbulls-blue (or other insoluble metalcyanides), etc. may be used in the course of a method as disclosedherein. It must be understood, that the named dyes and staining methodsshall be examples for the applicable methods, and that any other methodknown in the art may be applied to a method as disclosed herein. Thestaining procedures may produce chromogenic stains for light microscopicinspection or fluorescent stains for inspection under fluorescencemicroscopic conditions. In another embodiment of the present invention,radiation emitting procedures, procedures employing substances impairingthe transmission of radiation, or other contrast media for imaging ofthe cytological conditions in a sample (e.g., the generation of opticalimpression by means such as (micro-)autoradiographic or(micro-)radiographic picture generation) may be of use for a methodaccording to the present invention.

All the staining and imaging procedures may be used for analysis notonly in microscopic procedures, but also in automated analysisprocedures such flow cytometry, automated microscopic (computerized orcomputer aided) analysis, or any other method for analysis of stainedcytological specimens.

The analysis of the staining or imaging results of the differentprocedures may be performed in a single analysis step or in differentsubsequent steps. For example, the light microscopic inspection of aspecimen may be performed before or after fluorescence microsopicinspection of the specimen. In fluorescence microscopy, the analysis ofdifferent stains with different excitation wavelengths may be analyzedsimultaneously or subsequently. Other imaging methods may be employedsimultaneously or subsequently to the named procedures.

There may be various circumstances, under which combinations ofdifferent staining methods will be suitable. For example, in cases whereno satisfying cytological staining results may be achieved byimmuno-chemical staining, the additional application of generalcytological staining techniques may be suitable.

A sample according to the method of the present invention may compriseany sample comprising cells. Samples may comprise, e.g., secretions,smears, body fluids, and cell- or tissue-samples.

In one embodiment of the present invention, samples comprise cells ofthe anogenital tract, of the respiratory tract, or of the skin and itsappendages. In certain embodiments, the cells may be cells of theuterine cervix, the vagina, the vulva, the penis, the anus, the rectum,the bronchic tree, the lung, the peritoneum, the peritoneal space, thenaso-pharyngeal space, the oral cavity, or the skin. In certainembodiments of the present invention, the sample may be a histologicalsample, a biopsy, or a cytological sample such as, e.g., a smear, aswab, a wash, or a body fluid containing cells (sputum, a secretion,saliva, etc.). In certain embodiments of the present invention, thesamples may comprise cells infected by papilloma virus. The samples may,in certain embodiments, comprise cervical smears, bronchioalveolarlavages, etc.

In certain special embodiments of the present invention, the sample maybe prepared as a monolayer or thin layer preparation of a cytologicalspecimen. The respective methods for preparation of monolayer orthin-layer preparation in cytology are known to those of skill in theart. In one embodiment, the preparation may comprise, e.g., the ThinPreptechnology. Other methods comprise conventional smears, or methodemploying suspensions of cells for preparation of the cytologicalspecimens.

Preparation of a sample may comprise, e.g., obtaining a sample of atissue, of a body fluid, or of cells from a patient. According to thepresent invention, preparation of the sample may also comprise severalsteps of further preparations of the sample, such as preparation ofdissections, preparation of cell suspensions, spreading or applying thecells to be examined onto microscopic slides, preparation of tissuearrays, isolation of polypeptides or nucleic acids, preparation of solidphase fixed peptides or nucleic acids, or preparation of beads,membranes, or slides to which the molecules to be determined are coupledcovalently or non-covalently.

In certain embodiments of the present invention, the method may beperformed in an automated manner. The automation of the method may beachieved by automated staining and analysis of histological orcytological specimens on a solid surface by microscopic means. Inanother embodiment, the automation may comprise a flow-cytometricanalysis of the staining of cells in solution.

The dysplastic lesions to which the method according to the presentinvention may be applied comprise any dysplastic lesions characterizedby over-expression of INK4a gene products such as, e.g., p16^(INK4a) orp14ARF. In certain embodiments, those lesions are dysplasias associatedwith infections by papilloma viruses such as, e.g., HPV. In oneembodiment, the HPV may be a high risk HPV subtype such as HPV16, HPV18,HPV31, HPV 33, HPV35, HPV 39, HPV 45, HPV 51, HPV 52, HPV56, HPV 58, HPV59, HPV 66, HPV 68, etc. In one embodiment, the dysplastic lesions thatmay be detected according to the present invention comprise anogenitallesions, lesions of the respiratory tract, lesions of the head and theneck, or lesions of the skin and its appendages. Such lesions maycomprise dysplasias, e.g., of the anus or rectum, of the vulva, thevagina, the cervix or the penis, of the bronchic tree, the lung, theoral cavity, or the nasopharyngeal space. Another aspect of the presentinvention is a testing kit for performing the method according to thepresent invention. The kit may be, for example, a diagnostic kit or aresearch kit. A kit according to the present invention comprises atleast an agent suitable for detecting the INK4a gene-products.

Thus a kit according to present invention may comprise:

-   -   reagents for the detection of one or more INK4a gene-products;    -   reagents for the detection of one or more proliferation marker        gene-products;    -   reagents and buffers commonly used for carrying out the        detection reaction, such as buffers, reporters reactants (dyes,        etc.), carrier substances, and others;    -   INK4a gene product samples for carrying out a positive control        reaction; and    -   proliferation marker gene product samples for carrying out a        positive control reaction.

The reagents for the detection of the marker gene-products may includeany agent capable of binding to the marker gene-products. Such reagentsmay include proteins, polypeptides, nucleic acids, peptide nucleicacids, glycoproteins, proteoglycans, polysaccharides, or lipids.

The INK4a gene-product and proliferation marker gene product samples forcarrying out a positive control may comprise, for example, nucleic acidsin applicable form, such as solution or salt, peptides in applicableform, tissue section samples, or positive cells.

In a preferred embodiment of the invention, the detection of the markergene-products is carried out on the level of polypeptides. In thisembodiment, the binding agents may be, for example, antibodies specificfor the marker gene-products or fragments thereof.

In another embodiment of the test kit, the detection of the markergene-products is carried out on the nucleic acid level. In thisembodiment of the invention, the reagent for the detection may be, forexample, a nucleic acid probe or a primer reverse-complementary to saidmarker nucleic acids.

The present invention provides a method for the discrimination ofneoplastic and/or dysplastic and pre-neoplastic lesions, identifiable byassessment of the over-expression of p16^(INK4a), from other cellscells, which also detectably express p16^(INK4a), in the course ofhistological and/or cytological testing procedures. The method is basedon the detection of expressed gene-products of two or more INK4a geneproducts.

Thus the problem to be solved was to provide a method for discriminationbetween dysplastic cells and other cells lacking malignant growthpotential. The method may be applied to any stage of dysplasias and maybe especially useful in early stages, when cytological diagnosticmethods based on the p16^(INK4a) over-expression need furtherinformation for the identification of metaplastic cells. Furthermore thepresent invention provides a kit for performing the method according tothe present invention.

EXAMPLES

The following examples are given for the purpose of illustration onlyand are not intended to limit the scope of the invention disclosedherein. For the purpose of illustration, the methods disclosed hereinare exemplified using histological preparations. The histologicalexamples aid to judge whether the cells stained in one or the other wayare to be classified as dysplastic or metaplastic. The methods mayeasily be transferred to cytological specimens by altering the protocolin appropriate manner. These alterations are known to those of ordinaryskill in the art.

Example 1 Immunofluorescent Detection of the Over-Expression ofp16^(INK4a) and Ki67 in Samples of the Uterine Cervix (Double Staining)

Sections of formalin fixed, paraffin embedded tissue samples of thecervix uteri were immunofluorescent stained using antibodies specificfor p16^(INK4a) and Ki67. The tissue sections were rehydrated throughincubation in xylene and graded ethanol, and transferred to Aqua bidest.Antigen retrieval was carried out with 10 mM citrate buffer (pH 6.0) forp16^(INK4a) and Ki67. Therefore, the slides were heated in a waterbathfor 40 min at 95-98° C. The slides were cooled down to RT for 20minutes, and transferred to washing buffer (50 mM Tris-HCl, 150 mM NaCl,0.05% Tween 20/DakoCytomation: code no.: S3006).

To avoid non-specific binding of the secondary antibody (species: goat),the specimens were incubated with 10% goat serum for 30 min at RT.

The slides were then incubated with the primary antibodies, mouseanti-human p16^(INK4a) antibody (3.48 μg/ml) and rabbit anti Ki67 (1:25)for 30 min at RT, after which the slides were rinsed with washing bufferand placed in a fresh buffer bath for 5 min. Excess buffer was tappedoff, and each specimen was covered with 200 μl of the secondary reagentcontaining goat anti-mouse antibody, AlexaFluor®488 conjugated and goatanti-rabbit antibody, and Alexa Fluor®546 conjugated, and then incubatedfor 30 min at RT. Then slides were washed two times as before anddirectly mounted with a special mounting medium for fluorescence.

Microscopic examination of the slides revealed that cells immunoreactivewith p16^(INK4a) and also immunoreactive for Ki67 were only found insamples microscopically identified as samples of dysplastic lesions.Cells stained by the p16^(INK4a) specific reaction originating frommetaplasias were not stained by the reaction specific for Ki67.Microscopic inspection of the cell proliferation marker staining showedthat metaplastic cells over-expressing p16^(INK4a) were notimmunoreactive with the antibodies directed against Ki67. Samplescontaining dysplastic tissue areas, in contrast, comprised cells thatwere immunoreactive with Ki67 and with antibodies directed againstp16^(INK4a). So, in contrast to dysplasias in metaplasias, no cells weredouble stained using the Ki67 and p16^(INK4a) specific antibodies.

FIGS. 1-6 show the staining results for a severe dysplasia of the cervixuteri and for a squamous metaplasia using antibodies directed againstKi67 and against p16^(INK4a). Immunoreactivity for Ki67 rendered redfluorescence, immunoreactivity for p16^(INK4a) rendered greenfluorescence, and overlay of red and green fluorescence rendered yellowfluorescence. In the dysplastic specimen (FIGS. 1-3), many cells of thedysplasia showed nuclear positivity for Ki67, as well as positivity forp16^(INK4a), and thus gave rise to yellow fluorescence (see FIG. 3). Incontrast, in the metaplastic specimen (FIGS. 4-6), areas that positivelyexpressed p16^(INK4a) did not show any positive staining for Ki67,indicating lack of expression of the antigen in these areas. No doublestaining was observed in this specimen (FIG. 6) and thus, no cells inthe specimen gave rise to yellow fluorescence. These results show thatthe double staining of cells with reagents specific for Ki67 allowsdiscrimination of p16^(INK4a) over-expressing metaplasias fromdysplasias.

Example 2 Detection of Cells Co-Expressing p14ARF and mcm2 in Samples ofthe Uterine Cervix by In Situ Hybridization

Smears of the uterine cervix may be semi-quantitatively analysed for themRNA level of p16^(INK4a) and mcm2 in an in situ staining reaction. Thestaining reaction is performed as follows:

For rehydration, the spray-fixed smears are incubated in fresh 50% EtOHon a rocking device. The PEG film produced by the fixation procedure isremoved by intensive rinsing. Then, the smears are rinsed in Aquabidest. The smears are incubated with proteinese K (10 μg/ml in PBS) for10 min at 37° C. Then, the slides are transferred to washing buffer(PBS/0.1% Tween20) and finally, the area containing the cells issurrounded with a lipid-pencil.

The hybridization mixture is prepared by mixing 50 μl of ready-to-usehybridization buffer (DAKO A/S, Glostrup, Danmark) with about 5-10 μmolof the probes. The probes are biotin- and Digoxygenin-labelledoligonucleotides of sequences complememtary to the respective mRNAs.

The hybridization mixture is heated to 95° C. and afterwards,equilibrated to 37° C. After the boiling procedure, the smears areincubated with each 50 μl of the hybridization mixture for 4 hours at42° C. The samples are washed in excess volumes of the wash buffers twotimes in 2×SSC at 37° C. for 15 min, and once in 1×SSC at 37° C. for 15min. Then, the smears are rinsed two times at room temperature in 2×SSC.Following this washing procedure, the dissections are incubated for 30min with blocking buffer (NEN, blocking buffer) at room temperature,followed by a 1 hour incubation with a 1:100 diluted (in blockingbuffer, see above) Streptavidin-alkaline phosphatase and monoclonalmouse HRP-labeled anti-Digoxygenine antibodies (Molecular Probes). Thesmears are then washed 2 times in 1×PBS/0.1% Triton X-100 for 10 min atroom temperature, followed by one wash step with 1×PBS, 50 mM MgCl₂ (pH9.2) for 10 min at room temperature. Then the staining reaction isperformed with ELF 97 phosphate (Molecular Probes) for 10 sec to 7 minat room temperature. Excess substrate is washed 3 times with 1×PBS/0.1%Triton X-100 for 10 min at room temperature. In a second staining step,the section is incubated with Tyramides-Alexa-Fluor 594 for 10 sec to 7min. Excess substrate is washed 3 times with 1×PBS/0.1% Triton X-100 for10 min at room temperature. Finally the smears are dipped in H₂O_(dest.)and embedded with Fluorescence mounting medium (DakoCytomation). Thenthe stained dissections can be analysed by fluorescence microscopy.

Microscopic examination of the slides reveals that cells positive forexpression of p16^(INK4a) and mcm2 are only found in samples that aremicroscopically identified as samples of dysplastic lesions. Cellsstained by the p16^(INK4a) specific reaction that are identifiable asmetaplasias are not stained by the reaction specific for mcm2. Themicroscopic inspection of the mRNA hybridization shows that metaplasticcells over-expressing p16^(INK4a) do not significantly express mRNA ofmcm2. Dysplastic cells, in contrast, are stained by in situhybridization with probes specific for mcm2, and with probes directedagainst p16^(INK4a). So, in contrast to dysplastic cells, in metaplasticcells, no double staining using the mcm2 and p16^(INK4a) specific probesis observed. These results show that the double staining of cells withreagents specific for mcm2 allows discrimination of p16^(INK4a)over-expressing metaplasias from dysplasias.

Example 3 Immunofluorescent Detection of the Over-Expression ofp16^(INK4a) and Ki-S2 in Samples of the Uterine Cervix (Double Staining)

Merckofix®-fixed cytological samples (conventional smears andliquid-based cytology (ThinPreps®)) of the cervix uteri areimmunofluorescent stained using antibodies specific for p16^(INK4a) andKi-S2.

Conventional smears and liquid based cytological samples (ThinPreps®)are rehydrated in ethanol (50%) for 10 min and transferred in Aquabidest. Antigen retrieval is carried out with 10 mM citrate buffer (pH6.0) for p16^(INK4a) and Ki67. Therefore, the slides are heated in awaterbath for 40 min at 95°-98° C. The slides are cooled down to RT for20 minutes, transferred to washing buffer (50 mM Tris-HCl, 150 mM NaCl,0.05% Tween 20/DakoCytomation: code no.: S3006), and finally, thesamples are surrounded with a lipid-pencil.

To avoid non-specific binding of the secondary antibody (species: goat)the specimens are incubated with 10% goat serum for 30 min at RT.

The slides are then incubated with the primary antibodies, mouseanti-human p16^(INK4a) antibody (clone E6H4) (3.48 μg/ml) and rabbitanti-Ki-S2 (1:25) for 30 min at RT, and then the slides are rinsed withwashing buffer and placed in a fresh buffer bath for 5 min. Excessbuffer is tapped off, and the specimen is covered with 200 μl of thesecondary reagent containing goat anti-mouse antibody, AlexaFluor®488conjugated and goat anti rabbit antibody, and Alexa Fluor®546 conjugatedand then incubated for 30 min at RT. Then, slides are washed two timesas before and directly mounted with a special mounting medium forfluorescence. Microscopic examination of the slides reveals that cellsimmunoreactive with p16^(INK4a) and Ki-S2 may be identifiedmicroscopically as dysplastic cells. Cells stained by the p16^(INK4a)specific reaction, which are not stained by the reaction specific forKi-S2, may be classified by an experienced pathologist as either beingmetaplastic or of endometrial origin. Microscopic inspection of the cellproliferation marker staining shows that metaplastic cellsover-expressing p16^(INK4a) are not immunoreactive with the antibodiesdirected against Ki-S2. Dysplastic cells, in contrast, areimmunoreactive with Ki-S2 and with antibodies directed againstp16^(INK4a). So, in contrast to dysplastic cells, in metaplasias, nocells are double stained using the Ki-S2 and p16^(INK4a) specificantibodies. These results show that double staining of cells withreagents specific for Ki-S2 allows discrimination of p16^(INK4a)over-expressing metaplastic cells from dysplastic cells.

Example 4 Immunofluorescent Detection of the Over-Expression ofp16^(INK4a), Ki67 and PCNA in Bronchioalveolar-Lavage Samples ofIndividuals with Diagnosed Small Cell Lung Cancer (Double Staining)

Cells contained in bronchioalveolar lavage specimens of patients areprepared according to ThinPrep technology. Merckofix®-fixed cytologicalsamples of the lavages of patients diagnosed with small cell lung cancerare immunofluorescent stained using antibodies specific for p16^(INK4a),Ki67, and PCNA.

In this experiment, a procedure is used that does not discriminatebetween staining originating from immunoreactivity against the twoproliferation markers PCNA and Ki67. Within the context of the presentinvention, whether one or the other proliferation marker is expressed inthe cells must not necessarily be answered. The main question is whetherany proliferation marker is expressed. In the course of the experiments,a procedure is chosen that gives the same fluorescence signal as anindication of the presence of a proliferation marker. This procedure issuitable to improve sensitivity of the detection of the cellproliferation characteristics. As necessary, the procedure may beapplied so as to render one detectable signal for three, four, or evenmore marker molecules characteristic of cell proliferation. Analogously,the same may under certain circumstances be true for the INK4a geneexpression products. It must be understood that different stainingsignals for different proliferation marker molecules may be desirable.The procedures may be applied to the necessities of the respectiveexperiment.

The tissue sections are rehydrated through incubation in xylene andgraded ethanol, and then transferred to Aqua bidest. Conventional smearsand liquid-based cytological samples (ThinPreps®) are rehydrated inethanol (50%) for 10 min, and transferred in Aqua bidest. Antigenretrieval is carried out with 10 mM citrate buffer (pH 6.0) forp16^(INK4a), Ki67, and PCNA. Therefore, the slides are heated in awaterbath for 40 min at 95-98° C. The slides are cooled down to RT for20 minutes, transferred to washing buffer (50 mM Tris-HCl, 150 mM NaCl,0.05% Tween 20/DakoCytomation: code no.: S3006), and finally, thesamples are surrounded with a lipid-pencil. To avoid non-specificbinding of the secondary antibody (species: goat), the specimens areincubated with 10% goat serum for 30 min at RT.

The slides are then incubated with the primary antibodies, mouseanti-human p16^(INK4a) antibody (3.48 μg/ml), rabbit anti-Ki67, andrabbit anti-PCNA (each 1:25) for 30 min at RT. The slides are thenrinsed with washing buffer and placed in a fresh buffer bath for 5 min.Excess buffer is tapped off, and the specimen is covered with 200 μl ofthe secondary reagent (containing goat anti-mouse antibody,AlexaFluor®488 conjugated and goat anti rabbit antibody and, AlexaFluor®546 conjugated) and then incubated for 30 min at RT. Then slidesare washed two times as before and directly mounted with a specialmounting medium for fluorescence.

Microscopic examination of the slides reveals that cells immunoreactivewith p16^(INK4a) and also with Ki67 or PCNA may be identifiedmicroscopically as cells of small cell lung cancer. Cells stained by thep16^(INK4a)-specific reaction, originating from metaplasias, are notstained by the reaction specific for Ki67 and PCNA. The microscopicinspection of the cell proliferation marker staining shows thatmetaplastic cells over-expressing p16^(INK4a) are not immunoreactivewith antibodies directed against Ki67 and PCNA. Samples containingdysplastic cells, in contrast, comprise cells, that are immunoreactivewith Ki67/PCNA and with antibodies directed against p16^(INK4a). So, incontrast to dysplasias, in metaplasias, no cells may be triple-stainedusing the Ki67, PCNA, and p16^(INK4a) specific antibodies.

These results show that the triple staining of cells with reagentsspecific for Ki67/PCNA allows discrimination of p16^(INK4a)over-expressing non-dysplastic cells from dysplasias.

Example 5 Flow Cytometric Detection of Dysplastic Cells by SimultaneousDetection of mcm5 mRNA, p14ARF Protein, and Ki67 Protein in Cells ofCervical Origin

Cytological samples (Cell suspensions in PBS, pH 7.4) of the cervixuteri were fluorescent stained using antibodies specific for p14ARF andKi-67 and oligoprobes for mcm-5 and evaluated by three-color fluorescentFACS analysis.

Cells were centrifuged, the supernatant decanted and fixed andpermeabilized with 100 ml Permeafix (Ortho Diagnostic, Raitan, N.J.,USA) for 1 hour at room temperature. Cells were washed in sterile PBS,pH 7.4, pelleted, and re-suspended in 100 ml Permeafix for 1 hour atroom temperature. Cells were washed in sterile PBS, pH 7.4, pelleted,and re-suspended in sterile PBS. They were incubated with PE-conjugatedanti-p14ARF antibody and PE-Cy5-conjugated anti-Ki67 antibody for 1 h at+4° C. Cells were washed in sterile PBS, pH 7.4, pelleted, andre-suspended in 100 ml Permeafix for 30 min at room temperature. Cellswere washed in sterile PBS, pelleted by centrifugation, and then washedagain in 2× standard saline citrate (SSC). After centrifugation, thecell pellet was resuspended in hybridization solution (2×SSC, 30%formamide, sonicated salmon sperm, and yeast transfer DNA) containing500 ng of 5-carboxy-fluorescein double end-labelled, mcm5-specificoligonucleotides probes. The intercellular hybridization was performedat 42° C. for 1 hour, followed by successive washes in 2×SSC, 0.5%Triton X-100, and 1×SSC, 0.5% Triton X-100 at 42° C. The cells werere-suspended for analysis in PBS, pH 8.3, and analyzed on a flowcytometer (FACScan, Becton Dickinson, IS). For each analysis,30.000-100.000 gated events were collected. Data analysis was performedusing CellQuest (Becton Dickinson, IS). The flow cytometer analysisrevealed that cells immunoreactive with p14ARF and Ki-67, and/orreactive for the oligoprobes of mcm5, could be identified only insamples of patients with dysplastic lesions of the cervix. Samples fromwomen with no dysplastic lesions showed no concomitant staining ofp14ARF with Ki67 or mcm5.

These results show that the double-or-triple staining of cells withreagents specific for Ki67 and/or mcm5 allows discrimination of p14ARFover-expressing non-dysplastic cells from p14ARF over-expressingdysplastic cells.

Example 6 Immunoenzymatic Detection of the Over-Expression ofP16^(INK4a) and Ki67 in Histological Samples of the Uterine Cervix(Sequential Double Staining)

Sections of formalin-fixed, paraffin-embedded tissue samples of thecervix uteri were immunoenzymatically double-stained using antibodiesspecific for p16^(INK4a) and Ki67. The tissue sections were rehydratedthrough incubation in xylene and graded ethanol, and transferred to Aquabidest. Antigen retrieval was carried out with 10 mM citrate buffer (pH6.0) for p16^(INK4a) and Ki67. Therefore, the slides were heated in awaterbath for 40 min at 95-98° C. The slides were cooled down to RT for20 minutes and transferred to washing buffer (DakoCytomation).Endogenous peroxidase activities were blocked with 3% H₂O₂(DakoCytomation) for 5 min at RT. After washing the slides for 5 min atRT, they were incubated with the first primary antibody, mouseanti-human p16^(INK4a) antibody (MTM) for 30 min at RT, and were thenrinsed with washing buffer and placed in a fresh buffer bath for 5 min.Excess buffer was tapped off and each specimen was covered with 200 μlof the secondary reagent (EnVision goat antimouse—Peroxidase/DakoCytomation), and incubated for 30 min at RT. Thenslides were washed three times as before. For the chromogenicvisualization, DAB (DakoCytomation) was used by incubating the slideswith the substrate chromogen complex for 10 min at RT. The reaction wasstopped in deionized water, and the slides placed in wash buffer.

After washing, the slides were incubated with the second primaryantibody, rabbit anti-human Ki67 antibody (Dianova, clone Ab-3) for 30min at RT, and were then rinsed with washing buffer and placed in afresh buffer bath for 5 min. Excess buffer was tapped off, and eachspecimen was covered with 200 μl of the secondary reagent (goat antirabbit—Alkaline Phosphatase labeled/DakoCytomation) and incubated for 30min at RT. Then slides were washed three times as before. For thechromogenic visualization, FastRed (BioGenex) was used by incubating theslides with the substrate chromogen complex for 30 min at RT. Thereaction was stopped in deionized water. After counterstaining withhematoxylin (DakoCytomation) for 2 min at RT, the slides were incubatedin running tap water for 10 min at RT, and then mounted with aqueousmounting medium (Aquatex/MERCK).

The microscopic examination of the slides revealed that cellsimmunoreactive with p16^(INK4a) and Ki67 were found only in samples thatwere identified microscopically as samples of dysplastic lesions. Cellsstained by the p16^(INK4a)-specific reaction, originating frommetaplasias, were not stained by the reaction specific for Ki67.Microscopic inspection of the cell proliferation marker staining showedthat metaplastic cells over-expressing p16^(INK4a) were notimmunoreactive with antibodies directed against Ki67. Samples containingdysplastic tissue areas, in contrast, comprised cells that wereimmunoreactive with Ki67 and with antibodies directed againstp16^(INK4a). So, in contrast to dysplasias, in metaplasias, no cellswere double-stained using the Ki67 and p16^(INK4a) specific antibodies.

These results (FIG. 7) show that double staining of cells with reagentsspecific for Ki67 and p16^(INK4a) also produces a specificdouble-staining pattern in chromogenic staining procedures.

Example 7 Immunoenzymatic Detection of the Over-Expression ofp16^(INK4a) and Ki67 in Cytological Samples of the Uterine Cervix(Sequential Double Staining)

Merckofix® fixed cytological samples (conventional smears andliquid-based cytology (ThinPreps®)) of the cervix uteri wereimmunoenzymatically double-stained using antibodies specific forp16^(INK4a) and Ki67.

Conventional smears and liquid-based cytological samples were rehydratedin ethanol (50%) for 10 min at RT and transferred in Aqua bidest.Antigen retrieval was carried out with 10 mM citrate buffer (pH 6.0) forp16^(INK4a) and Ki67. Therefore, the slides were heated in a waterbathfor 40 min at 95-98° C. The slides were cooled down to RT for 20 minutesand transferred to washing buffer. Endogenous peroxidase activities wereblocked with 3% H₂O₂ for 5 min at RT. After washing, the slides wereincubated with the first primary antibody, mouse anti-human p16^(INK4a)antibody, for 30 min at RT, and were then rinsed with washing buffer andplaced in a fresh buffer bath for 5 min. Excess buffer was tapped off,and each specimen was covered with 200 μl of the secondary reagent(EnVision goat anti mouse—Peroxidase) and incubated for 30 min at RT.Then the slides were washed three times as before. For the chromogenicvisualization, DAB (DakoCytomation) was used by incubating the slideswith the substrate chromogen complex for 10 min at RT. The reaction wasstopped in deionized water, and the slides placed in wash buffer.

After washing, the slides were incubated with the second primaryantibody, rabbit anti-human Ki67 antibody (Dianova, clone Ab-3) for 30min at RT, and were then rinsed with washing buffer and placed in afresh buffer bath for 5 min. Excess buffer was tapped off, and eachspecimen was covered with 200 μl of the secondary reagent (goat antirabbit—Alkaline Phosphatase labeled/DakoCytomation) and incubated for 30min at RT. Then the slides were washed three times as before. For thechromogenic visualization, Fast Red (BioGenex) was used by incubatingthe slides with the substrate chromogen complex for 30 min at RT. Thereaction was stopped in deionized water.

After counterstaining with hematoxylin (DakoCytomation) for 2 min at RT,the slides were incubated in running tap water for 10 min at RT, andthen mounted with aqueous mounting medium (Aquatex/MERCK).

Microscopic examination of the slides revealed that cells immunoreactivewith p16^(INK4a) and Ki67 could be identified as dysplastic cells on thebasis of their morphology. Cells stained by the p16^(INK4a)-specificreaction originating from metaplasias were not stained by the reactionspecific for Ki67. The microscopic inspection of the cell proliferationmarker staining showed that metaplastic cells over-expressingp16^(INK4a) were not immunoreactive with the antibodies directed againstKi67. Dysplastic cells, in contrast, were immunoreactive with Ki67 andwith antibodies directed against p16^(INK4a). So, in contrast tometaplastic cells, in dysplastic cells, double-staining of single cellscould be produced using the Ki67 and p16^(INK4a) specific antibodies.

These results (FIG. 8) show that the double staining of cells withreagents specific for Ki67 and p16^(INK4a) also produces a specificdouble staining pattern in chromogenic staining procedures.

1-38. (canceled)
 39. A method of discriminating cervical pre-neoplastic,neoplastic, and/or dysplastic lesions from metaplastic lesions in asubject, comprising: (a) measuring an expression level of a high-riskHPV-associated nucleic acid marker in a sample collected from a subject,wherein expression at a detectable level of the high-risk HPV-associatednucleic acid marker is indicative of HPV infection, risk of neoplasia,and presence of high-risk HPV-associated nucleic acid marker; and (b)measuring an expression level of at least one protein marker selectedfrom a group consisting of p14 and p16 and at least one protein markerselected from a group consisting of MCM2, MCM3, MCM4, MCM5, MCM6, MCM7,HELAD1, CDC6, CDC7, Dbf4, CDC14, CDC45, MCM10, PCNA, POLD, Ki67, Ki-S5,and Ki-S2, in a sample from the subject, wherein the expression at adetectable level of at least one protein marker from each group isindicative of protein presence; wherein the expression at a detectablelevel of both high-risk HPV-associated nucleic acid marker in step (a)and at least one protein from each group in step (b) is indicative ofcervical pre-neoplastic, neoplastic and/or dysplastic lesion in thesubject.
 40. The method of claim 39, wherein the step of measuring anexpression level of at least one protein marker in each group in step(b) is performed in at least one single cell.
 41. The method of claim39, wherein the method is performed in a course of a cytological testingprocedure.
 42. The method of claim 39, wherein the step (b) comprisesmeasuring an expression level of p16 and Ki67.
 43. The method of claim39, wherein the HPV infection is a high-risk HPV infection selected fromthe group consisting of HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45,HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, and HPV68 infection.
 44. Themethod of claim 39, wherein measuring an expression level in step (b)comprises using at least one probe specifically recognizing at least oneprotein marker from each group.
 45. The method of claim 44, whereinmeasuring an expression level in step (b) comprises using at least oneantibody probe.
 46. The method of claim 45, wherein the at least oneantibody probe is recognized by a detectably labeled agent.
 47. Themethod of claim 46, wherein the detectably labeled agent is labeled witha label selected from the group consisting of a radioisotope, abioluminescent compound, a chemiluminescent compound, a fluorescentcompound, a metal chelate, and an enzyme.
 48. The method of claim 44,wherein measuring of an expression level in step (b) comprises acytochemical staining procedure rendering either a chromogenic or afluorescent signal.
 49. A method of discriminating cervicalpre-neoplastic, neoplastic, and/or dysplastic lesions from metaplasticlesions in a subject, comprising: (a) measuring an expression level of ahigh-risk HPV-associated nucleic acid marker in a sample collected froma subject, wherein expression at a detectable level of the high-riskHPV-associated nucleic acid marker is indicative of HPV infection, riskof neoplasia, and presence of high-risk HPV-associated nucleic acidmarker; and (b) measuring an expression level of at least one proteinmarker selected from a group consisting of p14 and p16 and at least oneprotein marker selected from a group consisting of MCM2, MCM3, MCM4,MCM5, MCM6, MCM7, HELAD1, CDC6, CDC7, Dbf4, CDC14, CDC45, MCM10, PCNA,POLD, Ki67, Ki-S5, and Ki-S2, in the sample from the subject, whereinthe expression at a detectable level of at least one protein marker fromeach group is indicative of protein presence; wherein the expression ata detectable level of both high-risk HPV-associated nucleic acid markerin step (a) and at least one protein from each group in step (b) in thesame sample is indicative of cervical pre-neoplastic, neoplastic and/ordysplastic lesion in the subject.
 50. The method of claim 49, whereinthe step of measuring an expression level of at least one protein markerin each group in step (b) is performed in at least one single cell. 51.The method of claim 49, wherein the method is performed in a course of acytological testing procedure.
 52. The method of claim 49, wherein thestep (b) comprises measuring an expression level of p16 and Ki67. 53.The method of claim 49, wherein the HPV infection is a high-risk HPVinfection selected from the group consisting of HPV16, HPV18, HPV31,HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66,and HPV68 infection.
 54. The method of claim 49, wherein measuring anexpression level in step (b) comprises using at least one probespecifically recognizing at least one protein marker from each group.55. The method of claim 54, wherein measuring an expression level instep (b) comprises using at least one antibody probe.
 56. The method ofclaim 55, wherein the at least one antibody probe is recognized by adetectably labeled agent.
 57. The method of claim 56, wherein thedetectably labeled agent is labeled with a label selected from the groupconsisting of a radioisotope, a bioluminescent compound, achemiluminescent compound, a fluorescent compound, a metal chelate, andan enzyme.
 58. The method of claim 54, wherein measuring of anexpression level in step (b) comprises a cytochemical staining procedurerendering either a chromogenic or a fluorescent signal.